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. 2015 Nov 19;13(9):1949–1964. doi: 10.1016/j.celrep.2015.10.056

Figure 2.

Figure 2

ADF/CFL1 Loss Causes Stress Fiber Accumulation

(A) Phase contrast images of keratinocytes (left panels) and fluorescent staining for filamentous actin (F-actin) (right panels).

(B) Squamous cell carcinoma cells (SCCs) were obtained from ADF−/− CFL1+/+ mice (ADF-null SCCs). Due to lethality, double knockout SCCs could not be propagated. The remaining isoform (CFL1) was transiently knocked down with siRNAs (siCFL1). Phase contrast images of ADF-null cells treated with non-targeting siRNAs (siNT) or siRNAs for CFL1 (siCFL1) are shown (left panels). F-actin is visualized by fluorescence in right panels.

(C) ADF-null SCCs expressing empty vector (pWZL) or exogenously expressed CFL1 (pWZL+CFL1wt) were treated with siNT or siRNAs targeting the UTR (siCFL1UTR) of CFL1 mRNA. Following siRNA treatment, cells were stained for F-actin.

(D) Percentage of ADF-null SCCs displaying aberrant actin accumulation following CFL1 depletion. 790 control and 615 CFL1 (total) depleted cells were quantified in four independent experiments. Result presented as mean ± SEM. ∗∗∗unpaired t test p value < 0.0001.

(E) ADF-null SCCs treated with siNT or siCFL1 for 48 hr were imaged every 30 s for 30 min. Representative montages of cells ruffling or blebbing their membranes are shown. White bars with arrowheads drawn perpendicular to membrane protrusions represent examples of areas for kymograph analysis. Movies of these cells are provided as Movies S1 and S2.

(F) Representative kymograph profiles from three siNT- and four siCFL1-treated cells. Profiles were plotted using normalized intensity values from 1-pixel-wide representative kymographs of membrane protrusions from siNT- and siCFL1-treated cells, composed parallel to protrusion direction over 30 min.

(G) Representative kymographs generated from cells shown in (E).

(H–J) Number of protrusions per 30 min, protrusion length, and time for a complete protrusion extension and retraction (protrusion persistence) are shown. Twenty cells with 83 and 20 cells with 34 protrusions were quantified following siNT or siCFL1 treatment, respectively (n = 4). Circles represent all individual values, and bars represent means ± SD; ∗∗∗Mann-Whitney p value ≤ 0.0002.

The scale bars represent 125 μm and 20 μm for phase and IF images, respectively. Nuclei were counterstained with DAPI in IF experiments. See also Figures S1B–S1H and S1J.